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September 22, 2019  |  

Evolution of selective-sequencing approaches for virus discovery and virome analysis.

Recent advances in sequencing technologies have transformed the field of virus discovery and virome analysis. Once mostly confined to the traditional Sanger sequencing based individual virus discovery, is now entirely replaced by high throughput sequencing (HTS) based virus metagenomics that can be used to characterize the nature and composition of entire viromes. To better harness the potential of HTS for the study of viromes, sample preparation methodologies use different approaches to exclude amplification of non-viral components that can overshadow low-titer viruses. These virus-sequence enrichment approaches mostly focus on the sample preparation methods, like enzymatic digestion of non-viral nucleic acids and size exclusion of non-viral constituents by column filtration, ultrafiltration or density gradient centrifugation. However, recently a new approach of virus-sequence enrichment called virome-capture sequencing, focused on the amplification or HTS library preparation stage, was developed to increase the ability of virome characterization. This new approach has the potential to further transform the field of virus discovery and virome analysis, but its technical complexity and sequence-dependence warrants further improvements. In this review we discuss the different methods, their applications and evolution, for selective sequencing based virome analysis and also propose refinements needed to harness the full potential of HTS for virome analysis. Copyright © 2017 Elsevier B.V. All rights reserved.


September 22, 2019  |  

Bacterial community structure in simultaneous nitrification, denitrification and organic matter removal process treating saline mustard tuber wastewater as revealed by 16S rRNA sequencing.

A simultaneous nitrification, denitrification and organic matter removal (SNDOR) process in sequencing batch biofilm reactor (SBBR) was established to treat saline mustard tuber wastewater (MTWW) in this study. An average COD removal efficiency of 86.48% and total nitrogen removal efficiency of 86.48% were achieved at 30gNaClL(-1) during 100days’ operation. The underlying mechanisms were investigated by PacBio SMRT DNA sequencing (V1-V9) to analyze the microbial community structures and its variation from low salinity at 10gNaClL(-1) to high salinity at 30gNaClL(-1). Results showed elevated salinity did not affect biological performance but reduced microbial diversity in SBBR, and halophilic bacteria gradually predominated by succession. Despite of high C/N, autotrophic ammonia-oxidizing bacteria (AOB) Nitrosomonas and ammonia-oxidizing archaea (AOA) Candidatus Nitrososphaera both contributed to ammonium oxidation. As salinity increasing, nitrite-oxidizing bacteria (NOB) were significantly inhibited, partial nitrification and denitrification (PND) process gradually contributed to nitrogen removal. Copyright © 2016 Elsevier Ltd. All rights reserved.


September 22, 2019  |  

Contrasting distribution patterns between aquatic and terrestrial Phytophthora species along a climatic gradient are linked to functional traits.

Diversity of microbial organisms is linked to global climatic gradients. The genus Phytophthora includes both aquatic and terrestrial plant pathogenic species that display a large variation of functional traits. The extent to which the physical environment (water or soil) modulates the interaction of microorganisms with climate is unknown. Here, we explored the main environmental drivers of diversity and functional trait composition of Phytophthora communities. Communities were obtained by a novel metabarcoding setup based on PacBio sequencing of river filtrates in 96 river sites along a geographical gradient. Species were classified as terrestrial or aquatic based on their phylogenetic clade. Overall, terrestrial and aquatic species showed contrasting patterns of diversity. For terrestrial species, precipitation was a stronger driver than temperature, and diversity and functional diversity decreased with decreasing temperature and precipitation. In cold and dry areas, the dominant species formed resistant structures and had a low optimum temperature. By contrast, for aquatic species, temperature and water chemistry were the strongest drivers, and diversity increased with decreasing temperature and precipitation. Within the same area, environmental filtering affected terrestrial species more strongly than aquatic species (20% versus 3% of the studied communities, respectively). Our results highlight the importance of functional traits and the physical environment in which microorganisms develop their life cycle when predicting their distribution under changing climatic conditions. Temperature and rainfall may be buffered differently by water and soil, and thus pose contrasting constrains to microbial assemblies.


September 22, 2019  |  

Soil microbial communities are shaped by plant-driven changes in resource availability during secondary succession.

Although we understand the ecological processes eliciting changes in plant community composition during secondary succession, we do not understand whether co-occurring changes in plant detritus shape saprotrophic microbial communities in soil. In this study, we investigated soil microbial composition and function across an old-field chronosequence ranging from 16 to 86 years following agricultural abandonment, as well as three forests representing potential late-successional ecosystems. Fungal and bacterial community composition was quantified from ribosomal DNA, and insight into the functional potential of the microbial community to decay plant litter was gained from shotgun metagenomics and extracellular enzyme assays. Accumulation of soil organic matter across the chronosequence exerted a positive and significant effect on fungal phylogenetic ß-diversity and the activity of extracellular enzymes with lignocellulolytic activity. In addition, the increasing abundance of lignin-rich C4 grasses was positively related to the composition of fungal genes with lignocellulolytic function, thereby linking plant community composition, litter biochemistry, and microbial community function. However, edaphic properties were the primary agent shaping bacterial communities, as bacterial ß-diversity and variation in functional gene composition displayed a significant and positive relationship to soil pH across the chronosequence. The late-successional forests were compositionally distinct from the oldest old fields, indicating that substantial changes occur in soil microbial communities as old fields give way to forests. Taken together, our observations demonstrate that plants govern the turnover of soil fungal communities and functional characteristics during secondary succession, due to the continual input of detritus and differences in litter biochemistry among plant species.


September 22, 2019  |  

Initial colonization, community assembly and ecosystem function: fungal colonist traits and litter biochemistry mediate decay rate.

Priority effects are an important ecological force shaping biotic communities and ecosystem processes, in which the establishment of early colonists alters the colonization success of later-arriving organisms via competitive exclusion and habitat modification. However, we do not understand which biotic and abiotic conditions lead to strong priority effects and lasting historical contingencies. Using saprotrophic fungi in a model leaf decomposition system, we investigated whether compositional and functional consequences of initial colonization were dependent on initial colonizer traits, resource availability or a combination thereof. To test these ideas, we factorially manipulated leaf litter biochemistry and initial fungal colonist identity, quantifying subsequent community composition, using neutral genetic markers, and community functional characteristics, including enzyme potential and leaf decay rates. During the first 3 months, initial colonist respiration rate and physiological capacity to degrade plant detritus were significant determinants of fungal community composition and leaf decay, indicating that rapid growth and lignolytic potential of early colonists contributed to altered trajectories of community assembly. Further, initial colonization on oak leaves generated increasingly divergent trajectories of fungal community composition and enzyme potential, indicating stronger initial colonizer effects on energy-poor substrates. Together, these observations provide evidence that initial colonization effects, and subsequent consequences on litter decay, are dependent upon substrate biochemistry and physiological traits within a regional species pool. Because microbial decay of plant detritus is important to global C storage, our results demonstrate that understanding the mechanisms by which initial conditions alter priority effects during community assembly may be key to understanding the drivers of ecosystem-level processes. © 2015 John Wiley & Sons Ltd.


September 22, 2019  |  

Single cell genomic study of Dehalococcoidetes species from deep-sea sediments of the Peruvian Margin.

The phylum Chloroflexi is one of the most frequently detected phyla in the subseafloor of the Pacific Ocean margins. Dehalogenating Chloroflexi (Dehalococcoidetes) was originally discovered as the key microorganisms mediating reductive dehalogenation via their key enzymes reductive dehalogenases (Rdh) as sole mode of energy conservation in terrestrial environments. The frequent detection of Dehalococcoidetes-related 16S rRNA and rdh genes in the marine subsurface implies a role for dissimilatory dehalorespiration in this environment; however, the two genes have never been linked to each other. To provide fundamental insights into the metabolism, genomic population structure and evolution of marine subsurface Dehalococcoidetes sp., we analyzed a non-contaminated deep-sea sediment core sample from the Peruvian Margin Ocean Drilling Program (ODP) site 1230, collected 7.3?m below the seafloor by a single cell genomic approach. We present for the first time single cell genomic data on three deep-sea Chloroflexi (Dsc) single cells from a marine subsurface environment. Two of the single cells were considered to be part of a local Dehalococcoidetes population and assembled together into a 1.38-Mb genome, which appears to be at least 85% complete. Despite a high degree of sequence-level similarity between the shared proteins in the Dsc and terrestrial Dehalococcoidetes, no evidence for catabolic reductive dehalogenation was found in Dsc. The genome content is however consistent with a strictly anaerobic organotrophic or lithotrophic lifestyle.


September 22, 2019  |  

Atmospheric N deposition increases bacterial laccase-like multicopper oxidases: implications for organic matter decay.

Anthropogenic release of biologically available nitrogen (N) has increased dramatically over the last 150 years, which can alter the processes controlling carbon (C) storage in terrestrial ecosystems. In a northern hardwood forest ecosystem located in Michigan in the United States, nearly 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. This change occurred concomitantly with compositional changes in Basidiomycete fungi and in Actinobacteria, as well as the downregulation of fungal lignocelluloytic genes. Recently, laccase-like multicopper oxidases (LMCOs) have been discovered among bacteria which can oxidize ß-O-4 linkages in phenolic compounds (e.g., lignin and humic compounds), resulting in the production of dissolved organic carbon (DOC). Here, we examined how nearly 2 decades of experimental N deposition has affected the abundance and composition of saprotrophic bacteria possessing LMCO genes. In our experiment, LMCO genes were more abundant in the forest floor under experimental N deposition whereas the abundances of bacteria and fungi were unchanged. Experimental N deposition also led to less-diverse, significantly altered bacterial and LMCO gene assemblages, with taxa implicated in organic matter decay (i.e., Actinobacteria, Proteobacteria) accounting for the majority of compositional changes. These results suggest that experimental N deposition favors bacteria in the forest floor that harbor the LMCO gene and represents a plausible mechanism by which anthropogenic N deposition has reduced decomposition, increased soil C storage, and accelerated phenolic DOC production in our field experiment. Our observations suggest that future rates of atmospheric N deposition could fundamentally alter the physiological potential of soil microbial communities. Copyright © 2014, American Society for Microbiology. All Rights Reserved.


September 22, 2019  |  

Long-term operation of microbial electrosynthesis systems improves acetate production by autotrophic microbiomes.

Microbial electrosynthesis is the biocathode-driven production of chemicals from CO2 and has the promise to be a sustainable, carbon-consuming technology. To date, microbial electrosynthesis of acetate, the first step in order to generate liquid fuels from CO2, has been characterized by low rates and yields. To improve performance, a previously established acetogenic biocathode was operated in semi-batch mode at a poised potential of -590 mV vs SHE for over 150 days beyond its initial development. Rates of acetate production reached a maximum of 17.25 mM day(-1) (1.04 g L(-1) d(-1)) with accumulation to 175 mM (10.5 g L(-1)) over 20 days. Hydrogen was also produced at high rates by the biocathode, reaching 100 mM d(-1) (0.2 g L(-1) d(-1)) and a total accumulation of 1164 mM (2.4 g L(-1)) over 20 days. Phylogenetic analysis of the active electrosynthetic microbiome revealed a similar community structure to what was observed during an earlier stage of development of the electroacetogenic microbiome. Acetobacterium spp. dominated the active microbial population on the cathodes. Also prevalent were Sulfurospirillum spp. and an unclassified Rhodobacteraceae. Taken together, these results demonstrate the stability, resilience, and improved performance of electrosynthetic biocathodes following long-term operation. Furthermore, sustained product formation at faster rates by a carbon-capturing microbiome is a key milestone addressed in this study that advances microbial electrosynthesis systems toward commercialization.


September 22, 2019  |  

A single-cell genome for Thiovulum sp.

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 µm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.


September 22, 2019  |  

Electrosynthesis of commodity chemicals by an autotrophic microbial community.

A microbial community originating from brewery waste produced methane, acetate, and hydrogen when selected on a granular graphite cathode poised at -590 mV versus the standard hydrogen electrode (SHE) with CO(2) as the only carbon source. This is the first report on the simultaneous electrosynthesis of these commodity chemicals and the first description of electroacetogenesis by a microbial community. Deep sequencing of the active community 16S rRNA revealed a dynamic microbial community composed of an invariant Archaea population of Methanobacterium spp. and a shifting Bacteria population. Acetobacterium spp. were the most abundant Bacteria on the cathode when acetogenesis dominated. Methane was generally the dominant product with rates increasing from <1 to 7 mM day(-1) (per cathode liquid volume) and was concomitantly produced with acetate and hydrogen. Acetogenesis increased to >4 mM day(-1) (accumulated to 28.5 mM over 12 days), and methanogenesis ceased following the addition of 2-bromoethanesulfonic acid. Traces of hydrogen accumulated during initial selection and subsequently accelerated to >11 mM day(-1) (versus 0.045 mM day(-1) abiotic production). The hypothesis of electrosynthetic biocatalysis occurring at the microbe-electrode interface was supported by a catalytic wave (midpoint potential of -460 mV versus SHE) in cyclic voltammetry scans of the biocathode, the lack of redox active components in the medium, and the generation of comparatively high amounts of products (even after medium exchange). In addition, the volumetric production rates of these three commodity chemicals are marked improvements for electrosynthesis, advancing the process toward economic feasibility.


September 22, 2019  |  

High-resolution phylogenetic microbial community profiling.

Over the past decade, high-throughput short-read 16S rRNA gene amplicon sequencing has eclipsed clone-dependent long-read Sanger sequencing for microbial community profiling. The transition to new technologies has provided more quantitative information at the expense of taxonomic resolution with implications for inferring metabolic traits in various ecosystems. We applied single-molecule real-time sequencing for microbial community profiling, generating full-length 16S rRNA gene sequences at high throughput, which we propose to name PhyloTags. We benchmarked and validated this approach using a defined microbial community. When further applied to samples from the water column of meromictic Sakinaw Lake, we show that while community structures at the phylum level are comparable between PhyloTags and Illumina V4 16S rRNA gene sequences (iTags), variance increases with community complexity at greater water depths. PhyloTags moreover allowed less ambiguous classification. Last, a platform-independent comparison of PhyloTags and in silico generated partial 16S rRNA gene sequences demonstrated significant differences in community structure and phylogenetic resolution across multiple taxonomic levels, including a severe underestimation in the abundance of specific microbial genera involved in nitrogen and methane cycling across the Lake’s water column. Thus, PhyloTags provide a reliable adjunct or alternative to cost-effective iTags, enabling more accurate phylogenetic resolution of microbial communities and predictions on their metabolic potential.


September 22, 2019  |  

Soil bacterial communities are shaped by temporal and environmental filtering: evidence from a long-term chronosequence.

Soil microbial communities are abundant, hyper-diverse and mediate global biogeochemical cycles, but we do not yet understand the processes mediating their assembly. Current hypothetical frameworks suggest temporal (e.g. dispersal limitation) and environmental (e.g. soil pH) filters shape microbial community composition; however, there is limited empirical evidence supporting this framework in the hyper-diverse soil environment, particularly at large spatial (i.e. regional to continental) and temporal (i.e. 100 to 1000 years) scales. Here, we present evidence from a long-term chronosequence (4000 years) that temporal and environmental filters do indeed shape soil bacterial community composition. Furthermore, nearly 20 years of environmental monitoring allowed us to control for potentially confounding environmental variation. Soil bacterial communities were phylogenetically distinct across the chronosequence. We determined that temporal and environmental factors accounted for significant portions of bacterial phylogenetic structure using distance-based linear models. Environmental factors together accounted for the majority of phylogenetic structure, namely, soil temperature (19%), pH (17%) and litter carbon:nitrogen (C:N; 17%). However, of all individual factors, time since deglaciation accounted for the greatest proportion of bacterial phylogenetic structure (20%). Taken together, our results provide empirical evidence that temporal and environmental filters act together to structure soil bacterial communities across large spatial and long-term temporal scales. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.


September 22, 2019  |  

Improved metagenome assemblies and taxonomic binning using long-read circular consensus sequence data.

DNA assembly is a core methodological step in metagenomic pipelines used to study the structure and function within microbial communities. Here we investigate the utility of Pacific Biosciences long and high accuracy circular consensus sequencing (CCS) reads for metagenomic projects. We compared the application and performance of both PacBio CCS and Illumina HiSeq data with assembly and taxonomic binning algorithms using metagenomic samples representing a complex microbial community. Eight SMRT cells produced approximately 94 Mb of CCS reads from a biogas reactor microbiome sample that averaged 1319 nt in length and 99.7% accuracy. CCS data assembly generated a comparative number of large contigs greater than 1?kb, to those assembled from a ~190x larger HiSeq dataset (~18 Gb) produced from the same sample (i.e approximately 62% of total contigs). Hybrid assemblies using PacBio CCS and HiSeq contigs produced improvements in assembly statistics, including an increase in the average contig length and number of large contigs. The incorporation of CCS data produced significant enhancements in taxonomic binning and genome reconstruction of two dominant phylotypes, which assembled and binned poorly using HiSeq data alone. Collectively these results illustrate the value of PacBio CCS reads in certain metagenomics applications.


September 22, 2019  |  

Atmospheric N deposition alters connectance, but not functional potential among saprotrophic bacterial communities.

The use of co-occurrence patterns to investigate interactions between micro-organisms has provided novel insight into organismal interactions within microbial communities. However, anthropogenic impacts on microbial co-occurrence patterns and ecosystem function remain an important gap in our ecological knowledge. In a northern hardwood forest ecosystem located in Michigan, USA, 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. This ecosystem-level response occurred concomitantly with compositional changes in saprophytic fungi and bacteria. Here, we investigated the influence of experimental N deposition on biotic interactions among forest floor bacterial assemblages by employing phylogenetic and molecular ecological network analysis. When compared to the ambient treatment, the forest floor bacterial community under experimental N deposition was less rich, more phylogenetically dispersed and exhibited a more clustered co-occurrence network topology. Together, our observations reveal the presence of increased biotic interactions among saprotrophic bacterial assemblages under future rates of N deposition. Moreover, they support the hypothesis that nearly two decades of experimental N deposition can modify the organization of microbial communities and provide further insight into why anthropogenic N deposition has reduced decomposition, increased soil C storage and accelerated phenolic DOC production in our field experiment. © 2015 John Wiley & Sons Ltd.


September 22, 2019  |  

Towards long-read metagenomics: complete assembly of three novel genomes from bacteria dependent on a diazotrophic cyanobacterium in a freshwater lake co-culture.

Here we report three complete bacterial genome assemblies from a PacBio shotgun metagenome of a co-culture from Upper Klamath Lake, OR. Genome annotations and culture conditions indicate these bacteria are dependent on carbon and nitrogen fixation from the cyanobacterium Aphanizomenon flos-aquae, whose genome was assembled to draft-quality. Due to their taxonomic novelty relative to previously sequenced bacteria, we have temporarily designated these bacteria as incertae sedis Hyphomonadaceae strain UKL13-1 (3,501,508 bp and 56.12% GC), incertae sedis Betaproteobacterium strain UKL13-2 (3,387,087 bp and 54.98% GC), and incertae sedis Bacteroidetes strain UKL13-3 (3,236,529 bp and 37.33% GC). Each genome consists of a single circular chromosome with no identified plasmids. When compared with binned Illumina assemblies of the same three genomes, there was ~7% discrepancy in total genome length. Gaps where Illumina assemblies broke were often due to repetitive elements. Within these missing sequences were essential genes and genes associated with a variety of functional categories. Annotated gene content reveals that both Proteobacteria are aerobic anoxygenic phototrophs, with Betaproteobacterium UKL13-2 potentially capable of phototrophic oxidation of sulfur compounds. Both proteobacterial genomes contain transporters suggesting they are scavenging fixed nitrogen from A. flos-aquae in the form of ammonium. Bacteroidetes UKL13-3 has few completely annotated biosynthetic pathways, and has a comparatively higher proportion of unannotated genes. The genomes were detected in only a few other freshwater metagenomes, suggesting that these bacteria are not ubiquitous in freshwater systems. Our results indicate that long-read sequencing is a viable method for sequencing dominant members from low-diversity microbial communities, and should be considered for environmental metagenomics when conditions meet these requirements.


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